Cow's milk allergy (CMA) is the most common food allergy in young children and is now likely to persist into adolescence and adulthood. Strict milk avoidance remains the only treatment for this allergy but accidental reactions, which are often severe, are extremely common, frequently due to the hidden presence of food allergens in processed foods and cross-contamination of foods. Recent studies from our institution and elsewhere have identified oral and sublingual allergen immunotherapy (OIT and SLIT) as promising approaches for treatment of CMA. Studies demonstrate that most children with IgE-mediated CMA can be desensitized to cow's milk protein, both safely and efficaciously. However, the specific mechanisms by which this treatment appears to work remain unknown, and questions remain to whether this increased tolerance to milk after treatment is permanent, or rather represents a transient desensitization which could place patients at significant risk of future reactions if milk was ever discontinued from their diet. Our long-range goal is to develop in vitro tests that help predict risks to adverse reactions to food as well as the safety/clinical efficacy of OIT/SLIT. The best chance of developing a routine test to make such predictions is best achieved with the versatility that serum/plasma provides. Our novel approach of using culture-derived basophils (CDBA) grown from adult stem cells should facilitate identification of plasma markers. Therefore, the proposal consists of 2 aims: Aim 1 involves analyzing plasma from CMA children undergoing SLIT and OIT for blocking activity by comparing pre and post specimens in passively sensitizing CDBA for subsequent challenge with milk allergen. The advantage of using CDBA is that they have never been exposed to immunoglobulins (IgE/IgG), which may otherwise confound the ability to detect blocking IgG4 antibody. Similar experiments will test pre- and post- OIT/SLIT plasma specimens for effects on plasmacytoid dendritic cells (pDCs), predicting that post-OIT/SLIT plasma (with greater IgG4 levels) will augment innate immune responses (TLR9) that are seemingly impaired in allergic individuals. Aim 2 explores the existing hypothesis that circulating allergen-IgE complexes are in plasma of CMA subjects and that these can be detected by inducing phenotypic/functional responses in CDBA and/or normal basophil. Plasma specimens from CMA subjects will be tested in flow-based assays for their ability to induce basophil spontaneous histamine release (SHR), the expression of markers (CD63/CD203c) linked to IgE-dependent activation as well as those (syk kinase) that are down regulated with prolonged Fc5RI cross-linking. These assays could result in new plasma-based tests useful in predicting whether one is at risk for adverse reactions and for predicting the clinical efficacy during successful OIT/SLIT. With food specific immunotherapy being potentially the most exciting area in the history of food allergy research, this project, with clinical and mechanistic endpoints should have immediate impact on the care of patients with food allergy.